CN107860924B - Application of the novel gamma delta T cells in preparation assessment AML curative effect reagent box - Google Patents
Application of the novel gamma delta T cells in preparation assessment AML curative effect reagent box Download PDFInfo
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- CN107860924B CN107860924B CN201710970962.3A CN201710970962A CN107860924B CN 107860924 B CN107860924 B CN 107860924B CN 201710970962 A CN201710970962 A CN 201710970962A CN 107860924 B CN107860924 B CN 107860924B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57426—Specifically defined cancers leukemia
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
Abstract
The present invention provides application of the novel gamma delta T cells subgroup in preparation prediction AML curative effect and prognosis kit；It is to find gamma delta T cells subgroup novel in AML peripheral blood in patients for the first time based on the present inventor, expression is related to the curative effect of AML patient and prognosis.When novel PD1+Foxp3+ gamma delta T cells Expression of Subsets ratio is high, a possibility that showing AML patient clinical weak curative effect, is larger.Novel gamma delta T cells Expression of Subsets ratio has important directive significance for the formulation of the Index for diagnosis and clinical treatment of AML patient, the present invention can provide more basic research data for the individualized treatment of AML patient, have broad application prospects in terms of prediction AML patient clinical curative effect and prognostic evaluation.
Description
Technical field
The invention belongs to field of biomedicine, in particular to novel gamma delta T cells subgroup is in preparation prediction AML curative effect and in advance
Application in kit afterwards.
Background technique
Acute myeloid leukaemia (acute myeloid leukemia, AML) is one group of evil for originating from candidate stem cell
Property Clonal disease, there is height heterogeneity, leukaemia cell stagnates due to losing the ability of differentiation and maturation in cell development
Different phase, and largely built up in marrow and other hematopoietic tissues, make normal hematopoiesis that obstacle occur, be in adult the most often
The type of leukemia seen.It mainly include at present inductive treatment and after treatment for the treatment of AML, in the past 30 years, although AML
Treatment achieves certain curative effect, for certain AML patients changed with bad chromosome or gene unconventionality due to can not be thorough
Intracorporal leukaemia cell is removed at bottom, is still faced with recurrence or drug resistance after alleviation.The AML patient of recurrent intractable is come at present
Say, there is no efficient treatment method, thus new therapeutic strategy up for it is further proposed that, to remove patient's minimal residual disease
Stove extends patient vitals, improves its life quality.
Current studies have shown that T cell adoptive immunotherapy is shown good application prospect for AML patient.People
Class periphery blood T cell can be divided into two subgroups of α β T cell and gamma delta T cells according to the difference of contained peptide chain.Gamma delta T cells only account for
The 1~10% of human peripheral blood T cell has non-principal histocompatibility complex (major histocompatibility
Complex, MHC) it is restricted identification antigen characteristic.In recent years more and more researches show that gamma delta T cells are with varied
Biological function, antineoplastic immune externally anti-infective in host, internally carry out immunosurveillance, immune defense, trnasplantion immunity and
The various aspects such as autoimmunity disease all play an important role.As that studies gamma delta T cells gradually gos deep into, it has been found that it is not
Only there is the powerful effect of activation immune response, under given conditions, while also there is immunoloregulation function.Traditional adjusting
Property T cell (regulatory T cells, Treg) be a kind of T cell with inhibitive ability of immunity and Immune anergy, tieing up
It plays an important role in terms of holding homeostasis；Research shows that under certain morbid states, the reduction of cell quantity or increase possibility
Take part in the occurrence and development of disease.Modulability gamma delta T cells (regulatory gamma delta T cells, gamma delta T reg) are newly to send out in recent years
The special subgroup of one of existing gamma delta T cells, expression specificity transcription factor plug winged-helix family transcriptional repressor intracellular
P3 (forkhead winged-helix family transcriptional repressor, Foxp3).Invention of the invention
People confirms (patent document that number of patent application is CN201610752852.5), the initial patient peripheral of AML in the research of early period
T cell receptor (T cell receptor, the TCR) pedigree of blood gamma delta T cells is distributed and difference has occurred in clonal expansion situation
The change of degree, possibility performed by different subgroups are not quite similar.It is still not relevant to gamma delta T cells subgroup effective at present
Predict the lab index of AML patient clinical curative effect and state evaluation；Industry is not also to PD-1 signal path way in AML patient
Whether diameter participates in regulation gamma delta T reg cell and carries out discussion research.The present invention be based on presently, there are the technical issues of and propose
New solution.
Summary of the invention
The primary purpose of the present invention is that the shortcomings that overcoming the prior art and deficiency, provide novel gamma delta T cells subgroup and are making
The application being ready for use in prediction AML curative effect and prognosis evaluation reagent kit.
Another object of the present invention is to provide a kind of prediction AML curative effect and the kits of prognosis evaluation.
A further object of the present invention is to provide a kind of for predicting the gamma delta T cells subgroup of AML curative effect and prognosis evaluation
Detection method.
The purpose of the invention is achieved by the following technical solution:
Novel gamma delta T cells subgroup, for predicting the application in AML curative effect and prognosis evaluation reagent kit, is to be based in preparation
The present inventor has found the treatment of gamma delta T cells Expression of Subsets situation and AML patient novel in AML peripheral blood in patients for the first time
It imitates related to prognosis.
The gamma delta T cells subgroup includes that Foxp3+ gamma delta T cells, PD-1+ gamma delta T cells and PD1+Foxp3+ gamma delta T are thin
One of born of the same parents or at least two.
When the described PD1+Foxp3+ gamma delta T cells expression ratio is high, a possibility that showing AML patient clinical weak curative effect
It is larger.
The PD1+Foxp3+ gamma delta T cells expression ratio height refers specifically to:
1. when the median of PD1+Foxp3+ gamma delta T cells proportion in total gamma delta T cells is 1.36%, AML illness
A possibility that it is larger；
2. when the median of PD1+Foxp3+ gamma delta T cells proportion in total gamma delta T cells is 0.32%；AML is alleviated
A possibility that it is larger；
3. being non-AML when the median of PD1+Foxp3+ gamma delta T cells proportion in total gamma delta T cells is 0.1%
A possibility that morbid state, is larger.
The AML illness includes AML initial, AML prognosis mala or AML recurrence.
A kind of kit for predicting AML curative effect and prognosis evaluation, the monoclonal antibody including following different fluorescent marker:
Anti- CD45 antibody, anti-cd 3 antibodies, anti-tcr γ anti-δ, anti-PD-1 antibody, the anti-PD-1 antibody isotype control Ab and
Anti- Foxp3 antibody.
The fluorescent marker of the anti-CD45 antibody is preferably V450.
The fluorescent marker of the anti-cd 3 antibodies is preferably Alexa Fluor 700.
The fluorescent marker of the anti-tcr γ anti-δ is preferably PE/Cy7.
The fluorescent marker of the isotype control Ab of the anti-PD-1 antibody or the anti-PD-1 antibody is preferably PE.
The fluorescent marker of the anti-Foxp3 antibody is preferably Alexa Fluor 647.
The kit further includes the corresponding isotype control Ab of monoclonal antibody.
The kit further includes the reagent for cell dyeing rupture of membranes, the examination for separating peripheral blood mononuclear cells
One of agent and phosphate buffer solution (PBS) or at least two.
The reagent for cell dyeing rupture of membranes includes cell dyeing buffer, fixation/rupture of membranes buffer, rupture of membranes buffering
One of liquid or at least two.
The fixation/rupture of membranes buffer is preferably that Foxp3 fixes/rupture of membranes buffer；The rupture of membranes buffer is preferred
For Foxp3 rupture of membranes buffer.
The reagent for separating peripheral blood mononuclear cells is preferably lymphocyte separation medium (Ficoll).
It is a kind of for predicting the detection method of the gamma delta T cells subgroup of AML curative effect and prognosis evaluation, include the following steps:
(1) peripheral blood sample to be measured is handled, single cell suspension is formed；Specifically, by peripheral blood to be measured according to routine side
Method processing, isolated lymphocyte suspension；
(2) monoclonal antibody for marking different fluorescence is added in the single cell suspension that step (1) obtains: anti-CD45 is anti-
Body, anti-cd 3 antibodies, anti-tcr γ anti-δ, anti-PD-1 antibody, the anti-PD-1 antibody isotype control Ab, mix gently
After be protected from light incubation；
(3) fixation/rupture of membranes buffer is added after washing cell, is protected from light incubation, washs cell again, after mixing well, from
The heart removes supernatant；
(4) cell is washed, rupture of membranes buffer is added, cells from light incubation is resuspended；
(5) supernatant is removed in centrifugation, and the anti-Foxp3 antibody after fluorescent marker is added is protected from light incubation；
(6) PBS resuspension cell is added after washing cell, machine testing on flow cytometer is described after obtaining fluorescent marker
Gamma delta T cells subgroup data；
(7) interpretation of result: carrying out statistical analysis to the expression ratio of the gamma delta T cells subgroup, when detecting PD1+
When Foxp3+ gamma delta T cells expression ratio is high, show that the prognosis mala possibility of AML patient is big.
The final concentration of lymphocyte suspension described in step (1) is preferably 1x106/100μL。
The additional proportion of anti-CD45 antibody described in step (2) be preferably every 100 μ L lymphocyte suspension proportion 3~
Anti- CD45 antibody described in 6 μ L；The lymphocyte suspension of further preferably every 100 μ L matches anti-CD45 antibody described in 5 μ L.
The additional proportion of anti-cd 3 antibodies described in step (2) is preferably the lymphocyte suspension proportion 3~6 of every 100 μ L
Anti-cd 3 antibodies described in μ L；The lymphocyte suspension of further preferably every 100 μ L matches anti-cd 3 antibodies described in 5 μ L.
The additional proportion of anti-tcr γ anti-δ described in step (2) is preferably the lymphocyte suspension proportion 3 of every 100 μ L
Anti-tcr γ anti-δ described in~6 μ L；The lymphocyte suspension of further preferably every 100 μ L matches anti-tcr γ described in 5 μ L
Anti-δ.
The additional proportion of the isotype control Ab of anti-PD-1 antibody described in step (2) or the anti-PD-1 antibody is excellent
The lymphocyte suspension for being selected as respectively every 100 μ L matches anti-PD-1 antibody or the anti-PD-1 antibody described in 3~6 μ L
Isotype control Ab；The lymphocyte suspension of further preferably every 100 μ L matches anti-PD-1 antibody or described described in 5 μ L
The isotype control Ab of anti-PD-1 antibody.
The concrete operations that incubation is protected from light described in step (2) are that incubation 30 minutes is protected from light at 4 DEG C.
The concrete operations that incubation is protected from light described in step (3) are that incubation 20 minutes is protected from light at 4 DEG C.
Washing described in step (3) is preferably washed with cell dyeing buffer.
The concrete operations that incubation is protected from light described in step (4) are that incubation 15 minutes is protected from light at 4 DEG C.
Washing described in step (4) is preferably washed with rupture of membranes buffer.
The concrete operations that incubation is protected from light described in step (5) are that incubation 30 minutes is protected from light at 4 DEG C.
Statistical analysis described in step (7) preferably carries out Pearson correlation analysis.
The expression ratio height of PD1+Foxp3+ gamma delta T cells described in step (7) refers specifically to:
1. when the median of PD1+Foxp3+ gamma delta T cells proportion in total gamma delta T cells is 1.36%, AML illness
A possibility that it is larger；
2. when the median of PD1+Foxp3+ gamma delta T cells proportion in total gamma delta T cells is 0.32%；AML is alleviated
A possibility that it is larger；
3. being non-AML when the median of PD1+Foxp3+ gamma delta T cells proportion in total gamma delta T cells is 0.1%
A possibility that morbid state, is larger.
The methods and applications are non-diagnostic purposes.
Although current existing program death receptor 1 (programmed death-1, PD-1) and programmed death receptor-
1 ligand -1 (programmed death-1ligand, PD-L1) signal path approach may promote the function of Treg cell and divide
The report of change.But it is thin whether the present inventor participates in regulation gamma delta T reg to PD-1 signal path approach in AML patient for the first time
This technological gap of born of the same parents has carried out research and inquirement, to provide the reality of more efficiently evaluation AML patient clinical curative effect and state
Test room index.Therefore the present invention combines clinical data, innovatively studies gamma delta T cells under AML state in detail by flow cytometry
Functional subclass and distribution are preferably to Leukemia Patients treated to efficiently obtain gamma delta T cells and its subgroup, can be
AML adoptive immunotherapy provides more scientific theories.
The present invention has the following advantages and effects with respect to the prior art:
1. the present inventor is had found for the first time in adult peripheral blood there is a kind of novel subgroup PD1+Foxp3+ gamma delta T cells,
The height situation of expression ratio and the morbid state of AML patient and lapse to certain correlation；It can be used as the prognosis of AML patient
The formulation of one of index, Index for diagnosis and clinical treatment for AML patient has important directive significance.
2., can be by the novel γ by the detection method the present invention provides the detection method of PD1+Foxp3+ gamma delta T cells
Delta T cells subgroup characterization and quantitative statistics, have broad prospects in terms of prediction AML patient clinical curative effect and prognostic evaluation, can
More basic research data are provided for AML individualized treatment.
Detailed description of the invention
Fig. 1 is the initial patient of AML and 3 class gamma delta T cells Functional subclass of healthy control group peripheral blood expression ratio fluidic cell
Art result analysis chart.
Fig. 2 is healthy control group and 3 class gamma delta T cells Functional subclass expression of the peripheral blood analysis of the initial patient of AML
Figure.
Fig. 3 is the correlation analysis knot of the expression ratio between 3 class gamma delta T cells Functional subclass of healthy control group peripheral blood
Fruit figure.
Fig. 4 is the correlation analysis knot of the expression ratio between the initial 3 class gamma delta T cells Functional subclass of peripheral blood in patients of AML
Fruit figure.
Fig. 5 is the analysis result figure of initial group of AML, the gentle system of solutions gamma delta T cells Functional subclass expression ratio of recurrence group.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Agents useful for same information is specific as follows in embodiment:
V450 marks mouse anti human CD45 (H130 is purchased from BD Pharmingen)；
Alexa Fluor 700 marks mouse anti human CD3 (UCHT1 is purchased from Biolegend)；
PE/Cy7 marks mouse anti human TCR γ δ (B1 is purchased from Biolegend)；
PE marks mouse anti human PD-1 (EH12.2H7 is purchased from Biolegend)；
Alexa Fluor 647 marks mouse anti human Foxp3 (206D is purchased from Biolegend)；
Cell dyeing buffer (cell staining buffer is purchased from Biolegend)；
Foxp3 fixes/rupture of membranes buffer (Foxp3Fix/Perm buffer is purchased from Biolegend)；
Foxp3 rupture of membranes buffer (Foxp3Perm buffer is purchased from Biolegend).
Embodiment 1
(1) it takes a blood sample under the premise of signing informed consent form with patient, all samples are taken from early morning limosis vein blood liver
Element is anticoagulant.AML initial 14 are collected, 4 Patients with Peripheral blood sample of patients with recurrent 5 and reduction of patient.It is collected simultaneously health adult's sample
This 15, which has obtained Ethics Committee, our unit and has passed through.AML patient clinical curative effect etc. is collected simultaneously to face
Bed data (as shown in table 1).
(2) separating peripheral blood mononuclear cells.Take lymphocyte separation medium (Ficoll, density 1.077) 4mL that 15mL is added
In centrifuge tube, the anticoagulation cirumferential blood sample suspension after dilution is laid on separating liquid, with horizontal centrifuge in 1500rpm
Centrifugation 15 minutes.It draws intermediate single stratum nucleare to be transferred in another 15mL centrifuge tube, adds appropriate 1 × PBS, gently blow and beat,
With 1000rpm centrifuge washing 10 minutes, supernatant is abandoned, 1 × PBS liquid is added and is counted after mixing to 2mL, and is washed with same method
It washs twice, by 1x106/ 100 μ L adjustment cell concentration is spare, obtains cell suspension.
(3) expression of Flow cytometry gamma delta T cells Functional subclass
3.1 need to prepare 2 streaming pipes with every sample, and 1 is set as to test tube, and 1 pipe is set as homotype pipe, and 100 μ are added in every pipe
Cell suspension made from L step (2).
3.2 are being separately added into each 5 μ L of corresponding surface molecular fluorescence antibody to test tube and homotype pipe, anti-including mouse
People V450-CD45, Alexa Fluor 700-CD3, PE/Cy7-TCR γ δ, FITC-TCR V δ 1, PerCp-TCR V δ 2；It is to be measured
5 μ L of PE-PD-1 is added in pipe, and corresponding 5 μ L of PE-PD-1 isotype control Ab is added in homotype pipe, and after mixing gently, 4 DEG C are protected from light and incubate
It educates 30 minutes.
3.3 are added cell staining buffer is washed cell 5 minutes with the revolving speed of 300g.
3.4 are added after 1mL Foxp3Fix/Perm buffer is mixed well to each streaming pipe and in 4 DEG C are protected from light incubation 20
Minute washs cell with cell staining buffer with the revolving speed of 300g and removes supernatant after five minutes.
3.5 wash cell with 1mL Foxp3Perm buffer again.Cell is resuspended with 1mL Foxp3Perm buffer
Incubation 15 minutes is protected from light in 4 DEG C.
Remove supernatant after 3.6 centrifugations, Alexa Fluor 647-Foxp3 antibody is added and is protected from light incubation 30 minutes in 4 DEG C, washes
After washing cell, it is resuspended after cell with 500 μ L PBS and obtains data, institute's total using flow type analyzer (BD Verse, USA) analysis
It is analyzed according to FlowJo Software.
(4) it by the expression combination AML patient clinical data of gamma delta T cells Functional subclass, is carried out with patient clinical curative effect
It analyzes (table 1), it is found that there is a kind of PD1+Foxp3+ gamma delta T cells (Fig. 1 and Fig. 2) in AML peripheral blood in patients.Using
Pearson analyzes PD-1 in each group gamma delta T cells+Subgroup, Foxp3+Subgroup and PD1+Foxp3+The correlation of Expression of Subsets ratio,
As a result the PD1 in healthy control group gamma delta T cells is prompted+Gamma delta T subgroup, Foxp3+Gamma delta T subgroup and PD1+Foxp3+Gamma delta T subgroup
It expresses without significant correlation (such as Fig. 3), and in the PD1 of the initial patient group of AML+Gamma delta T subgroup and PD1+Foxp3+Gamma delta T subgroup,
Foxp3+Gamma delta T subgroup and PD1+Foxp3+Positive correlation is presented in the expression ratio of gamma delta T subgroup, prompts patient Foxp3+γδT
Subset proportions obviously increase, and and PD1+Gamma delta T subgroup and PD1+Foxp3+The high proportion of gamma delta T subgroup is consistent, reflects patient γ
Immunosuppressive condition (such as Fig. 4) is presented in delta T cells；AML is initial and patients with recurrent in Foxp3+ gamma delta T cells, PD1+ gamma delta T cells
(Fig. 5) is obviously increased with PD1+Foxp3+ gamma delta T cells expression ratio.Ratio of the PD1+Foxp3+ gamma delta T cells under each state of AML
Example is in recurrence group > initial group > alleviation group.It can be seen from the above result that PD1+ gamma delta T cells and PD1+Foxp3+ gamma delta T in recurrence group
Cell expression obviously increases, may be related to the recurrent intractable of AML patient, and novel PD1+Foxp3+ gamma delta T cells in the present invention
Discovery provide more analysis assessments and basic research data for the individualized treatment of recurrent intractable patient.Above-mentioned experiment knot
Fruit shows there is important meaning in the assessment of prediction AML clinical efficacy and patient's states by detection gamma delta T cells Functional subclass
Justice.
Table 1AML patient clinical data
Note: F indicates women, and M indicates male.
Although diagnostic result in future and health status cannot be immediately arrived at from the above-mentioned expression ratio measured, in its conduct
Between as a result, can be used as one of the reference information of patient clinical therapeutic scheme formulation.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (4)
1. the kit of a kind of prediction AML patient clinical curative effect and prognosis evaluation, which is characterized in that including following different fluorescence
The monoclonal antibody of label:
Anti- CD45 antibody, anti-cd 3 antibodies, anti-tcr γ anti-δ, anti-PD-1 antibody, the Isotype control of the anti-PD-1 antibody are anti-
Body and anti-Foxp3 antibody.
2. the kit of prediction AML patient clinical curative effect according to claim 1 and prognosis evaluation, it is characterised in that:
The fluorescent marker of the anti-CD45 antibody is V450；
The fluorescent marker of the anti-cd 3 antibodies is Alexa Fluor 700；
The fluorescent marker of the anti-tcr γ anti-δ is PE/Cy7；
The fluorescent marker of the isotype control Ab of the anti-PD-1 antibody or the anti-PD-1 antibody is PE；
The fluorescent marker of the anti-Foxp3 antibody is Alexa Fluor 647.
3. the kit of prediction AML patient clinical curative effect according to claim 1 and prognosis evaluation, it is characterised in that:
The kit further includes the corresponding isotype control Ab of monoclonal antibody.
4. the kit of prediction AML patient clinical curative effect according to claim 1 and prognosis evaluation, it is characterised in that:
The kit further include the reagent for cell dyeing rupture of membranes, the reagent for separating peripheral blood mononuclear cells and
One of phosphate buffer solution or at least two.
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| CN111504886A (en) * | 2020-05-06 | 2020-08-07 | 西安交通大学 | Application of a group of molecules in preparation of auxiliary diagnosis reagent or kit for new coronary pneumonia |
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| CN109270265A (en) * | 2018-10-12 | 2019-01-25 | 东莞暨南大学研究院 | The lethal immune cell function assessment kit of human peripheral blood and appraisal procedure |
| CN109254148A (en) * | 2018-10-12 | 2019-01-22 | 东莞暨南大学研究院 | Human peripheral blood T cell immune function simplifies assessment kit and appraisal procedure |
| CN109781987B (en) * | 2019-01-09 | 2022-04-26 | 暨南大学 | Application of terminal effector T cell subset in preparation of kit for auxiliary evaluation of aplastic anemia disease degree |
| CN109752548B (en) * | 2019-02-01 | 2022-05-06 | 广州金域医学检验中心有限公司 | Combined reagent and system for evaluating prognosis of chronic lymphocytic leukemia |
| CN111621568A (en) * | 2020-06-23 | 2020-09-04 | 暨南大学 | Application of BRD4-PD-1 and/or BRD4-PD-L1 in preparation of AML prognosis prediction kit |
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